Collagen comprises a third of the protein in humans. Collagen abnormalities are associated with a wide variety of human diseases. The long-term objective of the proposed research is to obtain an atomic-level understanding of collagen structure and conformational stability. Each polypeptide chain of collagen is composed of repeats of the sequence: XaaYaaGly, where Xaa is often an L-proline (Pro) residue and Yaa is often a 4(R)-hydroxy L-proline (Hyp) residue. The hydroxyl groups of the Hyp residues contribute greatly to the conformational stability of triple-helical collagen. A long-standing paradigm had been that this stability arises from hydrogen bonds mediated by networks of bridging water molecules. In the prior funding period, this paradigm was disproved. Specifically, collagen in which Yaa is a 4(R)-fluoro-L-prolineresidue was shown to have much greater conformational stability than does collagen in which Yaa is a Hyp residue. The research proposed herein is designed to reveal why an electronegative atom at the 4(R) position enhances collagen stability as well as to test new hypotheses about collagen structure. This proposal has four specific aims. (1) Peptide synthesis. Defined mimics of triple-helical collagen containing non-natural residues will be synthesized. The hydroxyl group of endogenous Hyp residues will be modified with isologous electron-withdrawing groups. (2) Templated mimics. A new class of templated triple- helical collagen mimics will be synthesized, and these mimics will be used to assay for strand invasion (i.e., D-loop formation) by single strands. (3) Energetics. Circular dichroism spectroscopy and differential scanning calorimetry will be used to assess the conformational stabilities and folding rates of the collagen mimics. NMR spectroscopy will be used to determine the H/D fractionation factors of the solvent inaccessible, interstrand hydrogen bond in the collagen mimics. (4) Structure. NMR spectroscopy will be used to determine the ring pucker in the synthetic proline derivatives. Ab initio molecular orbital calculations will be used to enhance understanding of the chemical determinants of collagen stability. The results of the proposed research will provide new insights into the structure and stability of triple-helical collagen, and could ultimately lead to the creation of collagen mimics with important therapeutic applications.